Wednesday, February 15, 2012

Luciferin - Luciferase Experiment - Part I

I wanted to see the Luciferin - Luciferase reaction that produces bioluminescence and Mrs. Keel helped me to get this experiment going. She pointed me to a biological supplies store (Carolina Biological Supply) from where I got Sea Firefly lanterns (Cypridina hilgendorfii), gave me instructions on how to perform the experiment (how to separate out the luciferin and luciferase from the firefly lanterns), and allowed me to use her room to perform the experiment one morning before school. Thank you, Mrs. Keel!

Sea Firefly Lanterns

This short video clip shows the experiment that I performed: http://youtu.be/827Obd_Vm-s

I prepared two firefly lantern extracts, one of luciferin and the other of luciferase. I crushed some of the lanterns and placed them on an evaporation plate. I added distilled water to the crushed mixture and immediately a reaction started taking place. Luciferase acted upon luciferin and bioluminescence was produced. The luminescence slowly started dissipating as the luciferin in the extract got used up. When the luminescence completely disappeared, what I had left on the evaporation plate was a pure luciferase extract.

To create the pure luciferin extract, I added crushed firefly lanterns to boiling water. What this accomplished was to denature the luciferase. Luciferase does not automatically denature even when returned to room temperature.

I know have two separate containers, one with pure luciferase extract and the other with pure luciferin extract. Part II of my experiment will involve mixing these two together to see if they produce bioluminescence.

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Luciferase is an enzyme that catalyzes production of light from luciferin in the presence of Mg2+-ATP and oxygen. The reaction of this enzyme with luciferin, ATP, and O2 results in the emission of light. Luciferase activity can be inhibited by general anesthetics including isoflurane and ketamine/medetomidine thereby affecting the sensitivity of bioluminescence imaging. luciferase